Abstract
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.
Highlights
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions
We report the first use of a LOV domain as a fluorescent reporter in the anaerobic bacterium C. difficile
Whilst its relative small size (~13 kDa) makes it attractive as a reporter, its green fluorescence had been thought to limit its potential application in naturally autofluorescent bacteria, such as C. difficile
Summary
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions Their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Such as Light, Oxygen or Voltage sensing (LOV) domains have shown promising potential as real time probes in oxygen depleted situations[11,12,13,14,15]. The use of improved LOV (iLOV) or photostable iLOV (phiLOV) as fluorescent reporters has three main advantages over GFP as a reporter; phiLOV is smaller (~13 kDa vs ~25 kDa), fluorescence is stable over a greater pH range[23], and fluorescence is not dependent on molecular oxygen[24]
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